The WT1 gene (Wilms' tumor gene 1) has been identified as one of causative genes of Wilms' tumor that is a childhood renal tumor (Cell 60: 509, 1990, Nature 343: 774, 1990). WT1 gene encodes the transcription factor WT1, and WT1 plays an important role in many processes such as proliferation, differentiation and apoptosis of cells, and development of tissues (Int. Rev. Cytol. 181: 151, 1998). The WT1 gene was originally defined as a tumor suppressor gene. However, subsequent studies revealed that WT1 gene is highly expressed in leukemia and various solid cancers including lung cancer and breast cancer, and thus, indicating that WT1 gene rather exerts an oncogenic function promoting cancer growth. In addition, it was demonstrated that stimulation in vitro of peripheral blood mononuclear cells, which cells are positive to HLA-A*0201 or HLA-A*2402, with WT1-derived peptides induces the peptide-specific cytotoxic T-lymphocytes (CTLs), and the CTLs kill leukemia or solid tumor cells endogenously expressing WT1. These results demonstrated that WT1 is a promising target molecule of cancer immunotherapy (Int. J. Hematol 76: 127, 2002).
There have been known methods for determining in vitro antigen peptide-specific CTLs, including HLA monomer method, HLA dimer method and HLA tetramer method (Science 274: 94, 1996), HLA pentamer method and ELISPOT method (J. Immunol. Methods 110: 29, 1988), realtime RT-PCR technique (J. Immunol. Methods 210: 195, 1997), limiting dilution method (Br. J. Cancer 77: 1907, 1998), and the like. HLA-tetramer is prepared by biotinylating a complex (HLA monomer) formed by association of HLA α-chain and β2-microglobulin with a peptide, and allowing the monomer to bind to fluorescence-labeled avidin for tetramerization. The frequencies of CTLs can be measured by staining peptide-specific CTLs with HLA tetramers and analyzing by flow cytometry. Measurement of CTL frequencies by HLA monomer method, HLA dimer method and HLA pentamer method can be carried out on the basis of the same principle.
CTLs not stimulated with vaccine are referred to as “CTL precursor cells”. It is considered that the higher the frequency of existence of CTL precursor cells specific for a given cancer antigen, the more efficiently induced specific CTLs can be, when the said antigen is administered as a cancer vaccine, which makes it easier to attain clinical response of cancer vaccine therapy. In other words, if a patient showing high frequency of existence regarding CTL precursor cells specific for a given caner antigen is selected prior to vaccination, it would be possible to treat more effectively by the use of said cancer antigen.
Frequency of CTL precursor cells were measured using HLA tetramer and peripheral blood mononuclear cells (PBMC) of melanoma patients and reported in several papers; however, they show that the frequency of CTL precursor cells specific for tumor antigen peptide is low (J. Immunother. 24: 66, 2001, Hum. Gene. Ther. 13: 569, 2002). From these results, it has been regarded that the frequency of existence of CTL precursor cells specific for antigen is generally low and that it is difficult to select patients suitable for a cancer vaccine on the basis of the frequency of CTL precursor cells as an indicator.